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Bcl-X(L) specifically activates Bak to induce swelling and restructuring of the endoplasmic reticulum.

Klee M, Pimentel-Muiños FX - J. Cell Biol. (2005)

Bottom Line: Experiments with Bak and Bax-deficient mouse embryonic fibroblasts show that endogenous Bak mediates the effect, whereas Bax is mainly irrelevant.These results reveal a previously unidentified role of Bak in regulating reticular conformation.Because this activity is absent in Bax, it constitutes one of the first examples of functional divergence between the two multidomain homologues.

View Article: PubMed Central - PubMed

Affiliation: Centro de Investigación del Cáncer, Universidad de Salamanca-CSIC, Salamanca, 37007 Spain.

ABSTRACT
Bcl-2 family members Bak and Bax constitute a mitochondrial gateway for multiple death pathways. Both proteins are also present in the endoplasmic reticulum where they control apoptosis through the regulation of calcium levels. We show here that reticular Bak has the additional capacity of modulating the structure of this organelle. Coexpression of Bak and Bcl-X(L) provokes extensive swelling and vacuolization of reticular cisternae. A Bak version lacking the BH3 domain suffices to induce this phenotype, and reticular targeting of this mutant retains the activity. Expression of upstream BH3-only activators in similar conditions recapitulates ER swelling and vacuolization if ryanodine receptor calcium channel activity is inhibited. Experiments with Bak and Bax-deficient mouse embryonic fibroblasts show that endogenous Bak mediates the effect, whereas Bax is mainly irrelevant. These results reveal a previously unidentified role of Bak in regulating reticular conformation. Because this activity is absent in Bax, it constitutes one of the first examples of functional divergence between the two multidomain homologues.

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Effect of endogenous Bak and Bax in cytoplasmic vacuolization. (A) Bak-deficient MEFs are unable to generate ER swelling in response to coexpression of BimEL or tBid with Bcl-XL in the presence of dantrolene. The different MEFs were transfected with a mix of the indicated plasmids and a plasmid expressing GFP. Transfection efficiencies ranged between 20 and 30%. Dantrolene (50 μM) was added 1 h after transfection to all wells. Z-VAD.fmk (100 μM) was also added 1 h after transfection to reduce background death. 48 h later the proportion of GFP-expressing cells showing cytoplasmic vacuolization was determined as in Fig. 1 B. Shown is one representative example of three independent experiments. (B) Expression of Bak-ΔBH3 induces cytoplasmic vacuolization irrespective of the endogenous expression of Bak or Bax. MEFs were cotransfected with the indicated plasmids and a plasmid expressing GFP. Transfection, treatments and scoring were done as in A.
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fig8: Effect of endogenous Bak and Bax in cytoplasmic vacuolization. (A) Bak-deficient MEFs are unable to generate ER swelling in response to coexpression of BimEL or tBid with Bcl-XL in the presence of dantrolene. The different MEFs were transfected with a mix of the indicated plasmids and a plasmid expressing GFP. Transfection efficiencies ranged between 20 and 30%. Dantrolene (50 μM) was added 1 h after transfection to all wells. Z-VAD.fmk (100 μM) was also added 1 h after transfection to reduce background death. 48 h later the proportion of GFP-expressing cells showing cytoplasmic vacuolization was determined as in Fig. 1 B. Shown is one representative example of three independent experiments. (B) Expression of Bak-ΔBH3 induces cytoplasmic vacuolization irrespective of the endogenous expression of Bak or Bax. MEFs were cotransfected with the indicated plasmids and a plasmid expressing GFP. Transfection, treatments and scoring were done as in A.

Mentions: To test this possibility, we turned to bak−/− and bax−/− MEFs. Reminiscent of previous results with 293T cells (Fig. 7 A), transfection of BimEL or tBid in combination with Bcl-XL induced wild-type MEFs to show cytoplasmic vacuolization in the presence of dantrolene (Fig. 8 A). Vacuolae had a reticular origin as shown by experiments using cotransfected erRFP (unpublished data). Although this phenotype was basically unchanged in Bax-deficient MEFs (Fig. 8 A), the absence of Bak almost completely abolished it (Fig. 8 A). In addition, expression of Bak-ΔBH3 induced a comparable extent of cytoplasmic vacuolization in all tested MEFs (Fig. 8 B), indicating that endogenous full-length Bak is unnecessary for the activity of Bak-ΔBH3. All these results point to a model where upstream ER-remodelling signals are entirely transmitted through endogenous Bak, whereas Bax is for the most part inactive.


Bcl-X(L) specifically activates Bak to induce swelling and restructuring of the endoplasmic reticulum.

Klee M, Pimentel-Muiños FX - J. Cell Biol. (2005)

Effect of endogenous Bak and Bax in cytoplasmic vacuolization. (A) Bak-deficient MEFs are unable to generate ER swelling in response to coexpression of BimEL or tBid with Bcl-XL in the presence of dantrolene. The different MEFs were transfected with a mix of the indicated plasmids and a plasmid expressing GFP. Transfection efficiencies ranged between 20 and 30%. Dantrolene (50 μM) was added 1 h after transfection to all wells. Z-VAD.fmk (100 μM) was also added 1 h after transfection to reduce background death. 48 h later the proportion of GFP-expressing cells showing cytoplasmic vacuolization was determined as in Fig. 1 B. Shown is one representative example of three independent experiments. (B) Expression of Bak-ΔBH3 induces cytoplasmic vacuolization irrespective of the endogenous expression of Bak or Bax. MEFs were cotransfected with the indicated plasmids and a plasmid expressing GFP. Transfection, treatments and scoring were done as in A.
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fig8: Effect of endogenous Bak and Bax in cytoplasmic vacuolization. (A) Bak-deficient MEFs are unable to generate ER swelling in response to coexpression of BimEL or tBid with Bcl-XL in the presence of dantrolene. The different MEFs were transfected with a mix of the indicated plasmids and a plasmid expressing GFP. Transfection efficiencies ranged between 20 and 30%. Dantrolene (50 μM) was added 1 h after transfection to all wells. Z-VAD.fmk (100 μM) was also added 1 h after transfection to reduce background death. 48 h later the proportion of GFP-expressing cells showing cytoplasmic vacuolization was determined as in Fig. 1 B. Shown is one representative example of three independent experiments. (B) Expression of Bak-ΔBH3 induces cytoplasmic vacuolization irrespective of the endogenous expression of Bak or Bax. MEFs were cotransfected with the indicated plasmids and a plasmid expressing GFP. Transfection, treatments and scoring were done as in A.
Mentions: To test this possibility, we turned to bak−/− and bax−/− MEFs. Reminiscent of previous results with 293T cells (Fig. 7 A), transfection of BimEL or tBid in combination with Bcl-XL induced wild-type MEFs to show cytoplasmic vacuolization in the presence of dantrolene (Fig. 8 A). Vacuolae had a reticular origin as shown by experiments using cotransfected erRFP (unpublished data). Although this phenotype was basically unchanged in Bax-deficient MEFs (Fig. 8 A), the absence of Bak almost completely abolished it (Fig. 8 A). In addition, expression of Bak-ΔBH3 induced a comparable extent of cytoplasmic vacuolization in all tested MEFs (Fig. 8 B), indicating that endogenous full-length Bak is unnecessary for the activity of Bak-ΔBH3. All these results point to a model where upstream ER-remodelling signals are entirely transmitted through endogenous Bak, whereas Bax is for the most part inactive.

Bottom Line: Experiments with Bak and Bax-deficient mouse embryonic fibroblasts show that endogenous Bak mediates the effect, whereas Bax is mainly irrelevant.These results reveal a previously unidentified role of Bak in regulating reticular conformation.Because this activity is absent in Bax, it constitutes one of the first examples of functional divergence between the two multidomain homologues.

View Article: PubMed Central - PubMed

Affiliation: Centro de Investigación del Cáncer, Universidad de Salamanca-CSIC, Salamanca, 37007 Spain.

ABSTRACT
Bcl-2 family members Bak and Bax constitute a mitochondrial gateway for multiple death pathways. Both proteins are also present in the endoplasmic reticulum where they control apoptosis through the regulation of calcium levels. We show here that reticular Bak has the additional capacity of modulating the structure of this organelle. Coexpression of Bak and Bcl-X(L) provokes extensive swelling and vacuolization of reticular cisternae. A Bak version lacking the BH3 domain suffices to induce this phenotype, and reticular targeting of this mutant retains the activity. Expression of upstream BH3-only activators in similar conditions recapitulates ER swelling and vacuolization if ryanodine receptor calcium channel activity is inhibited. Experiments with Bak and Bax-deficient mouse embryonic fibroblasts show that endogenous Bak mediates the effect, whereas Bax is mainly irrelevant. These results reveal a previously unidentified role of Bak in regulating reticular conformation. Because this activity is absent in Bax, it constitutes one of the first examples of functional divergence between the two multidomain homologues.

Show MeSH
Related in: MedlinePlus